Hydrochloride salt form for ezh2 inhibition

ABSTRACT

Provided herein are novel solid forms of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide hydrochloride, and related compositions and methods.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/000,539, filed Jun. 5, 2018 (now allowed), which is a divisionalapplication of U.S. application Ser. No. 15/029,848, filed Apr. 15, 2016(now U.S. Pat. No. 10,040,782), which is a U.S. National Phaseapplication filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2014/060724, filed Oct. 15, 2014, which claims priority to,and the benefit of, the U.S. Provisional Application No. 61/891,786filed Oct. 16, 2013, the contents of each of which are incorporated byreference in their entireties.

TECHNICAL FIELD

This disclosure relates to solid crystalline forms ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride, and related compositions and methods.

BACKGROUND OF THE INVENTION

More than 1.6 million people are estimated to be diagnosed with cancerin 2013. For example, one of the most common types of cancer in women isbreast cancer, and this disease is responsible for one of the highestfatality rates of all cancers affecting females. The current treatmentof breast cancer is limited to total, or partial, mastectomy, radiationtherapy, or chemotherapy. More than 232,340 cancer cases in 2013 will bebreast cancer, which will result in an estimated 40,030 deaths. See,Siegel et al., CA: Cancer J. Clin. 2013; 63:11-30.

A number of cancer deaths are caused by blood cancers includingleukemias, myelomas, and lymphomas. In 2013, almost 80,000 cancer caseswill be lymphomas, estimated to result in over 20,000 deaths.

Radiation therapy, chemotherapy, and surgery are the primary methods ofcancer treatment. However, these therapies are most successful only whenthe cancer is detected at an early stage. Once cancer reachesinvasive/metastatic stages, lines of invading cells or metastasizingcells can escape detection, thus resulting in relapses, which requiresthe use of therapy that is highly toxic. At this point, both the cancercells and the patient's unaffected cells are exposed to the toxictherapy, resulting with, among other complications, a weakening of theimmune system. As such, there remains a need in the art for new methodsfor treating cancer, such as breast cancer or lymphoma, in a patient.

SUMMARY OF THE INVENTION

Accordingly, provided herein are novel solid forms (e.g., crystallineforms) ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride:

One embodiment of the invention is directed to Polymorph C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride . In one embodiment, Polymorph C is substantially free ofimpurities, meaning there is not a significant amount of impuritiespresent in the sample of Polymorph C. In another embodiment, Polymorph Cis a crystalline solid substantially free of amorphousN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(or any of its amorphous mono- or multi-HCl forms). The skilled artisanunderstands that a solid sample of Polymorph C may also includePolymorph A, Polymorph B, and/or amorphousN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(or any of its amorphous mono- or multi-HCl forms).

Polymorph C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride can be defined according to its X-ray powder diffractionpattern. Accordingly, in one embodiment, Polymorph C exhibits an X-raypowder diffraction pattern having characteristic peaks expressed indegrees 2-theta (+/−0.2) at 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and30.30.

In one embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having peaks with 2-theta values substantially in accordancewith FIG. 3. In another embodiment, Polymorph C exhibits an X-ray powderdiffraction pattern having peaks with 2-theta values substantially inaccordance with Table 3.

Polymorph C can also be defined according to its differential scanningcalorimetry thermogram. In one embodiment, the polymorph exhibits adifferential scanning calorimetry thermogram showing a primary endothermexpressed in units of ° C. at a temperature of 228 +/−5° C. In anotherembodiment, Polymorph C exhibits a differential scanning calorimetrythermogram substantially in accordance with the lowermost plot shown inFIG. 4 (i.e., “Polymorph C” plot).

Another aspect of the invention relates to the preparation of PolymorphC using a method comprising combiningN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidewith hydrochloric acid.

Another aspect of the invention relates to the preparation of, providedherein is a method of recrystallizing Polymorph C, which comprises thefollowing steps: (a) dissolving Polymorph C in a first solvent to obtaina first solution, and (b) adding a second solvent to the first solution,such that said polymorph is recrystallized.

In still another aspect, provided herein is a pharmaceutical compositioncomprising Polymorph C, and optionally a pharmaceutically acceptablecarrier or diluent. In one embodiment, the pharmaceutical compositioncomprises Polymorph C and a pharmaceutically acceptable carrier ordiluent.

Also provided herein is a method of treating an EZH2-mediated cancercomprising administering to a subject in need thereof a therapeuticallyeffective amount of Polymorph C, or a pharmaceutical compositionthereof. A variety of EZH2-mediated cancers may be treated withPolymorph C, including non-Hodgkin's lymphoma, B cell lymphoma includingdiffuse large B cell lymphoma (DLBCL), follicular lymphoma, or solidtumors including breast cancer.

In another aspect, provided herein is a method of inhibiting the histonemethyltransferase activity of EZH2 in a subject in need thereofcomprising administering to the subject an effective amount of PolymorphC, or a pharmaceutical composition thereof.

In still another aspect, provided herein is a method of inhibiting thehistone methyltransferase activity of EZH2 in vitro comprisingadministering Polymorph C or a pharmaceutical composition thereof.

Also provided herein is the use of Polymorph C, or a pharmaceuticalcomposition thereof, for the preparation of a medicament for thetreatment of an EZH2-mediated cancer in a subject in need thereof.

Another aspect of this invention is a method of treating or preventingan EZH2-mediated disorder. The method includes administering to asubject in need thereof a therapeutically effective amount of one ormore polymorphs disclosed herein. The EZH2-mediated disorder is adisease, disorder, or condition that is mediated at least in part by theactivity of EZH2. In one embodiment, the EZH2-mediated disorder isrelated to an increased EZH2 activity. In one embodiment, the increasedEZH2 activity is due to a mutation in the SET domain of EZH2. In oneembodiment, the mutation is at Y641, A677, or A687, or a combinationthereof. In one embodiment, the EZH2 mutation increases trimethylationof Lys27 of histone H3 (H3-K27). In one embodiment, the EZH2-mediateddisorder is a cancer. The EZH2-mediated cancer may be lymphoma, leukemiaor melanoma, for example, diffuse large B-cell lymphoma (DLBCL),non-Hodgkin's lymphoma (NHL), follicular lymphoma, chronic myelogenousleukemia (CML), acute myeloid leukemia, acute lymphocytic leukemia,mixed lineage leukemia, or myelodysplastic syndromes (MDS). In oneembodiment the EZH2-mediated cancer may be a malignant rhabdoid tumor orINI1-defecient tumor. The histologic diagnosis of malignant rhabdoidtumor depends on identification of characteristic rhabdoid cells (largecells with eccentrically located nuclei and abundant, eosinophiliccytoplasm) and immunohistochemistry with antibodies to vimentin, keratinand epithelial membrane antigen. In most malignant rhabdoid tumors, theSMARCB1/INI1 gene, located in chromosome band 22q11.2, is inactivated bydeletions and/or mutations. In one embodiment, the malignant rhabdoidtumors may be INI1-defecient tumor.

Unless otherwise stated, any description of a method of treatmentincludes uses of the polymorphs to provide such treatment or prophylaxisas is described in the specification, as well as uses of the polymorphsto prepare a medicament to treat or prevent such condition. Thetreatment includes treatment of human or non-human animals includingrodents and other disease models.

Further, the polymorphs or methods described herein may be used forresearch (e.g., studying epigenetic enzymes) and other non-therapeuticpurposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a representative X-ray powder diffraction pattern ofPolymorph A.

FIG. 2 depicts a representative X-ray powder diffraction pattern ofPolymorph B.

FIG. 3 depicts a representative X-ray powder diffraction pattern ofPolymorph C.

FIG. 4 depicts differential scanning calorimetry (DSC) data forPolymorphs A, B and C.

FIG. 5 depicts dynamic vapor sorption (DVS) data for Polymorph C.

FIG. 6 depicts DVS data for Polymorph A.

FIG. 7 depicts DSC data for Polymorph A.

DETAILED DESCRIPTION OF THE INVENTION

The solid form (e.g., crystal state) of a compound may be important whenthe compound is used for pharmaceutical purposes. Compared with anamorphous solid, the solid physical properties of a crystalline compoundmay change from one solid form to another, which may impact itssuitability for pharmaceutical use. In addition, different solid formsof a crystalline compound may incorporate different types and/ordifferent amounts of impurities. Different solid forms of a compound mayalso have different chemical stability upon exposure to heat, lightand/or moisture (e.g., atmospheric moisture) over a period of time, ordifferent rates of dissolution. There remains a need for solidcrystalline forms ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidethat are not hygroscopic, and that exhibit improved chemical stabilityfor use in drug substance and drug product development.

Provided herein are novel crystalline forms ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride:

Described herein are polymorphic forms A, B and C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride (also referred to herein respectively as “Polymorph A”,“Polymorph B” and “Polymorph C”).

As used herein, “Compound I” refers toN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide.The hydrochloride (i.e., hydrochloride salt) of Compound I may be usedto inhibit the histone methyltransferase activity of EZH2, either in asubject or in vitro. The hydrochloride of Compound I may also be used totreat EZH2-mediated cancer in a subject in need thereof.

Compound I can be protonated at one or more of its basic sites, such asthe morpholine, disubstituted aniline, and/or pyridone moieties. Thecompound may be protonated at any basic site. Without being limited tothe following, it is believed that Compound I is protonated at thenitrogen of the morpholino substituent, providing a hydrochloride ofCompound I having the following structure:

If there is any discrepancy as to the identity of Polymorph C as between(i) the above structure and (ii) the compound identified by the data ofFIGS. 3, 5 and the lowest plot depicted in FIG. 4, the latter (i.e.,Figures of (ii)) shall control.

The monohydrochloride drawn in the preceding paragraph can be referredto as“4-((3′-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumchloride.” The monohydrochloride salt ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidecan be produced in a highly crystalline form, which is useful in thepreparation of pharmaceutical formulations, and will improve generalhandling, manipulation, and storage of the drug compound. In a preferredembodiment, the crystalline form of the hydrochloride salt of Compound Iis in a form referred to as “Polymorph C.” As described herein,Polymorph C exhibits physical properties that can be exploited in orderto obtain new pharmacological properties, and that may be utilized indrug substance and drug product development.

The ability of a substance to exist in more than one crystal form isdefined as polymorphism; the different crystal forms of a particularsubstance are referred to as “polymorphs” of one another. In general,polymorphism is affected by the ability of a molecule of a substance (orits salt or hydrate) to change its conformation or to form differentintermolecular or intra-molecular interactions, (e.g., differenthydrogen bond configurations), which is reflected in different atomicarrangements in the crystal lattices of different polymorphs. Incontrast, the overall external form of a substance is known as“morphology,” which refers to the external shape of the crystal and theplanes present, without reference to the internal structure. Aparticular crystalline polymorph can display different morphology basedon different conditions, such as, for example, growth rate, stirring,and the presence of impurities.

The different polymorphs of a substance may possess different energiesof the crystal lattice and, thus, in solid state they can show differentphysical properties such as form, density, melting point, color,stability, solubility, dissolution rate, etc., which can, in turn,effect the stability, dissolution rate and/or bioavailability of a givenpolymorph and its suitability for use as a pharmaceutical and inpharmaceutical compositions.

Polymorph C has a number of advantageous physical properties over itsfree base form, as well as other salts of the free base. In particular,Polymorph C has low hygroscopicity compared to other salt forms ofCompound I. More particularly, Polymorph C has low hygroscopicitycompared to Polymorph A (i.e., another polymorph form ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride) (see, e.g., FIGS. 5 and 6). For consistency with drugformulation (e.g., tableting), it is generally required that thepolymorphic form of the active pharmaceutical ingredient (API) compoundbe minimally hygroscopic. Drug forms that are highly hygroscopic mayalso be unstable, as the drug form's dissolution rate (and otherphysico-chemical properties) may change as it is stored in settings withvarying humidity. Also, hygroscopicity can impact large-scale handlingand manufacturing of a compound, as it can be difficult to determine thetrue weight of a hygroscopic active agent when preparing apharmaceutical composition comprising that agent. For example, in largescale tableting or other medicinal formulating preparations, highlyhygroscopic compounds can result in batch manufacturing inconsistencycreating clinical and/or prescribing difficulties. Polymorph C has a lowhygoscopicity compared to other salt forms of Compound I. As such, itmay be stored over appreciable periods or conditions (e.g., relativehumidity conditions), and not suffer from detrimental formulatingchanges.

In certain embodiments, Polymorph C is identifiable on the basis ofcharacteristic peaks in an X-ray powder diffraction analysis. X-raypowder diffraction pattern, also referred to as XRPD pattern, is ascientific technique involving the scattering of x-rays by crystalatoms, producing a diffraction pattern that yields information about thestructure of the crystal. In certain embodiments, Polymorph C exhibitsan X-ray powder diffraction pattern having from two (2) to seven (7)characteristic peaks expressed in degrees 2-theta at 17.53, 18.66,21.14, 22.22, 23.46, 27.72 and 30.30.

The skilled artisan recognizes that some variation is associated with2-theta measurements. Typically, 2-theta values may vary from ±0.1 to±0.2. Such slight variation can be caused, for example, by samplepreparation and other experimental factors. The skilled artisanappreciates that such variation in values are greatest with low 2-thetavalues, and least with high 2-theta values. The skilled artisanrecognizes that different instruments may provide substantially the sameXRPD pattern, even though the 2-theta values vary somewhat. Moreover,the skilled artisan appreciates that the same instrument may providesubstantially the same XRPD pattern for the same or different sampleseven though the XRPD of the respectively collected XRPD patterns varyslightly in the 2-theta values.

The skilled artisan also appreciates that XRPD patterns of the samesample (taken on the same or different instruments) may exhibitvariations in peak intensity at the different 2-theta values. Theskilled artisan also appreciates that XRPD patterns of different samplesof the same polymorph (taken on the same or different instruments) mayalso exhibit variations in peak intensity at the different 2-thetavalues. XRPD patterns can be substantially the same pattern even thoughthey have corresponding 2-theta signals that vary in their peakintensities.

In one embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having two or more characteristic peaks expressed in degrees2-theta (+/−0.2) at 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and 30.30.In another embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having three or more characteristic peaks expressed in degrees2-theta (+/−0.2) at 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and 30.30.In another embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having four or more characteristic peaks expressed in degrees2-theta (+/−0.2) at 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and 30.30.In another embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having characteristic peaks expressed in degrees 2-theta(+/−0.2) at 17.53, 21.14, 23.46 and 27.72.

In a particular embodiment, Polymorph C exhibits an X-ray powderdiffraction pattern having at least eight characteristic peaks expressedin degrees 2-theta (+/−0.2), selected from the group consisting of10.08, 10.94, 16.58, 17.12, 17.53, 18.34, 18.66, 20.50, 21.14, 21.92,22.22, 23.46, 26.22, 26.60, 27.72, and 30.30. In another particularembodiment, Polymorph C exhibits an X-ray powder diffraction patternhaving at least nine characteristic peaks expressed in degrees 2-theta(+/−0.2), selected from the group consisting of 10.08, 10.94, 16.58,17.12, 17.53, 18.34, 18.66, 20.50, 21.14, 21.92, 22.22, 23.46, 26.22,26.60, 27.72, and 30.30.

In one embodiment, Polymorph C exhibits an X-ray powder diffractionpattern having a characteristic peaks expressed in degrees 2-theta(+/−0.2) at 27.72.

Pharmaceutical compositions comprising Polymorph C can be identified bycomparison of the compositions′ X-ray powder diffraction patterns to anX-ray powder diffraction pattern of Polymorph C. It will be appreciatedthat pharmaceutical compositions comprising Polymorph C may exhibitnon-identical X-ray powder diffraction patterns that are substantiallythe same pattern as compared to FIG. 3. Observed slight differences inXRPD patterns may be attributed to the aforementioned factors, includingthe presence of other impurities in the sample.

In other embodiments of the invention, Polymorph C is identifiable onthe basis of a characteristic peak observed in a differential scanningcalorimetry thermogram. Differential scanning calorimetry, or DSC, is athermoanalytical technique in which the difference in the amount of heatrequired to increase the temperature of a sample and reference ismeasured as a function of temperature. In one embodiment, Polymorph Cexhibits a differential scanning calorimetry thermogram showing acharacteristic peak expressed in units of ° C. with an onset temperatureof about 230+/−5° C. In another embodiment, Polymorph C exhibits adifferential scanning calorimetry thermogram showing a characteristicprimary endotherm expressed in units of ° C. at a temperature of about228+/−5° C. In another embodiment, Polymorph C exhibits a differentialscanning calorimetry thermogram substantially in accordance with FIG. 4.

In another embodiment of the invention, provided herein is Polymorph Ccharacterized as a solid form ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride, wherein the solid form undergoes a weight increase ofless than 1.5% upon increasing relative humidity from 5.0% to 95.0%. Inanother embodiment, Polymorph C is characterized as having a dynamicvapor sorption profile that is substantially in accordance with FIG. 5.

In certain embodiments, a sample of Polymorph C may contain impurities.Non-limiting examples of impurities include other polymorph forms, orresidual organic and inorganic molecules such as related impurities(e.g., intermediates used to make Polymorph C or fragments thereof),solvents, water or salts. In one embodiment, a sample of Polymorph C issubstantially free from impurities, meaning that no significant amountof impurities are present. In another embodiment, a sample of PolymorphC contains less than 10% weight by weight (wt/wt) total impurities. Inanother embodiment, a sample of Polymorph C contains less than 5% wt/wttotal impurities. In another embodiment, a sample of Polymorph Ccontains less than 2% wt/wt total impurities. In another embodiment, asample of Polymorph C contains less than 1% wt/wt total impurities. Inyet another embodiment, a sample of Polymorph C contains less than 0.1%wt/wt total impurities.

In certain embodiments, a sample of Polymorph C is a crystalline solidsubstantially free of amorphous Compound I (or any of its amorphousmono- or multi-HCl forms). As used herein, the term “substantially freeof amorphous Compound I” means that the compound contains no significantamount of amorphous Compound I (or any of its amorphous mono- ormulti-HCl forms). In another embodiment, a sample of crystallineCompound I comprises Polymorph C substantially free of Polymorph Aand/or B. As used herein, the term “substantially free of Polymorph Aand/or B” means that a sample of crystalline Compound I hydrochloridecontains no significant amount of Polymorph A and/or B. In certainembodiments, at least about 90% by weight of a sample is Polymorph C,with only 10% being Polymorph A and/or B and/or amorphous Compound I (orany of its amorphous mono- or multi-HCl forms). In certain embodiments,at least about 95% by weight of a sample is Polymorph C, with only 5%being Polymorph A and/or B and/or amorphous Compound I (or any of itsamorphous mono- or multi-HCl forms). In still other embodiments of theinvention, at least about 98% by weight of a sample is Polymorph C, withonly 2% by weight being Polymorph A and/or B and/or amorphous Compound I(or any of its amorphous mono- or multi-HCl forms). In still otherembodiments of the invention, at least about 99% by weight of a sampleis Polymorph C, with only 1% by weight being Polymorph A and/or B and/oramorphous Compound I (or any of its amorphous mono- or multi-HCl forms).In still other embodiments of the invention, at least about 99.5% byweight of a sample is Polymorph C, with only 0.5% by weight beingPolymorph A and/or B and/or amorphous Compound I (or any of itsamorphous mono- or multi-HCl forms). In still other embodiments of theinvention, at least about 99.9% by weight of a sample is Polymorph C,with only 0.1% by weight being Polymorph A and/or B and/or amorphousCompound I (or any of its amorphous mono- or multi-HCl forms).

Polymorph C may occur as any reasonable tautomer, or a mixture ofreasonable tautomers. As used herein, “tautomer” refers to one of two ormore structural isomers that exist in equilibrium and are readilyconverted from one isomeric form to another. Examples include keto-enoltautomers, such as acetone/propen-2-ol, and the like. Polymorph C mayhave one or more tautomers and therefore include various isomers, i.e.,pyridin-2(1H)-one and the corresponding pyridin-2-ol. All such isomericforms of these compounds are expressly included in the presentinvention.

Preparation of Polymorphs

General techniques for making polymorphs are understood by the skilledartisan. Conventionally, a salt form is prepared by combining insolution the free base compound and an acid containing the anion of thesalt form desired, and then isolating the solid salt product from thereaction solution (e.g., by crystallization, precipitation, evaporation,etc.). Other salt-forming techniques may be employed.

Once a polymorph is prepared, it may be recrystallized, using the samesolvent (or solvents) that were used to prepare the polymorph, or adifferent solvent (or solvents), to produce a composition that hasincreased crystallinity. In general, polymorphs may be recrystallized bydissolving the polymorph in one or more solvents, optionally heating,followed by an optional cooling step, and then isolating the crystalstructure, through, e.g., a filtering step. After the polymorph isinitially dissolved in the first solvent (or combination of solvents),an additional, different solvent may be added at any point in theprocess (before or after heating, before or after cooling, etc.) toproduce the desired crystal structure. For example, a first solvent maybe used to dissolve the polymorph compound, and then a second solvent(e.g., an anti-solvent) may be added to cause the polymorph toprecipitate from solution.

Non-limiting examples of solvents that may be used for therecrystallization of polymorphs are as follows: methanol, ethanol, ethylacetate, methyl tert-butyl ether, water, isopropyl alcohol,tetrahydrofuran, acetone, acetonitrile, and 2-methyltetrahydrofuran, aswell as combinations thereof. Non-limiting examples of solventcombinations that are useful for the recrystallization of polymorphs are(solvent and anti-solvent, wherein water can be added to the firstsolvent to aid in dissolving the polymorph): methanol/water and ethylacetate, isopropyl alcohol/water and ethyl acetate,tetrahydrofuran/water and ethyl acetate, acetone and ethyl acetate,acetonitrile/water and ethyl acetate, ethanol/water and methyltert-butyl ether, isopropyl alcohol/water and methyl tert-butyl ether,ethanol/water and tetrahydrofuran, isopropyl alcohol/water and acetone,and ethanol/water and ethyl acetate. In particular embodiments, thesolvent combinations may be ethanol/water and ethyl acetate, methanoland ethyl acetate, and ethanol and ethyl acetate.

In one aspect, provided herein is a method of preparing Polymorph C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride comprising combiningN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidewith hydrochloric acid.

In one embodiment, the method of making Polymorph C comprises the steps:

a) DissolvingN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′ -(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide in a first solvent to obtain a solution;

b) Combining hydrochloric acid with the solution;

c) Combining a second solvent with the solution;

d) Precipitating or crystallizing Polymorph C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidemonohydrochloride from the solution; and

e) Collecting Polymorph C ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide monohydrochloride.

In one embodiment of the method, the first solvent comprises ethanol. Inanother embodiment, the hydrochloric acid is in a concentrated aqueoussolution. In still another embodiment, the second solvent comprisesethyl acetate. In other embodiments, one or more of the solutions ofsteps a), b) or c) is heated.

In an embodiment, water is added to the first solvent to aid indissolving the polymorph.

In a particular embodiment of the method of making Polymorph C, asuspension ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(about 1 equivalent) in ethanol (about 1 volume) is heated, and treatedwith hydrochloric acid (about 1 equivalent). The mixture is stirred atelevated temperature, and is then treated with ethyl acetate (about 2volumes). The resulting mixture is stirred at elevated temperature andis then slowly cooled to room temperature. The resulting precipitate isfiltered, washed with ethyl acetate and dried to give Polymorph C.

In another aspect, provided herein is a method of recrystallizingPolymorph C, which comprises the following steps: (a) dissolvingPolymorph C in a first solvent to obtain a first solution, and (b)adding a second solvent, such that said polymorph is recrystallized. Inone embodiment, the method comprises (a) dissolving Polymorph C inethanol, (b) heating the mixture, (c) adding ethyl acetate to themixture, forming a precipitate comprising said polymorph, and filteringthe precipitate such that said polymorph is recrystallized. In oneembodiment, the first solvent is ethanol, and the second solvent isethyl acetate. In another embodiment, the first solvent is ethanol andwater, and the second solvent is ethyl acetate. In another embodiment,the first solvent is methanol, and the second solvent is ethyl acetate.In some embodiments, the method further comprises heating the firstsolution prior to adding the second solvent.

In another aspect, provided herein is Polymorph B ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidehydrochloride. In one embodiment, Polymorph B exhibits an X-ray powderdiffraction pattern substantially in accordance with FIG. 2. In anotherembodiment, Polymorph B exhibits an X-ray powder diffraction patternsubstantially in accordance with Table 2. In another embodiment,Polymorph B exhibits a differential scanning calorimetry thermogramhaving an onset temperature expressed in units of ° C. at a temperatureof 105+/−5° C. In another embodiment, Polymorph B exhibits a DSCthermogram substantially in accordance with FIG. 4. In anotherembodiment, Polymorph B exhibits a DSC thermogram substantially inaccordance with Table 4.

Pharmaceutical Compositions

In another aspect, provided herein is a pharmaceutical compositioncomprising polymorphs of the present invention (e.g., Polymorph C), andoptionally a pharmaceutically acceptable carrier or diluent. Alsoprovided herein is a pharmaceutical composition comprising polymorphs ofthe present invention (e.g., Polymorph C) and a pharmaceuticallyacceptable carrier or diluent.

The term “pharmaceutical composition” includes preparations suitable foradministration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1% to 99.9% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The polymorphs described herein (e.g., Polymorph C) may be combined witha pharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques. As used herein, “pharmaceuticallyacceptable carrier” may include any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition, its use is contemplated to be within thescope of this invention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatine; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil, sesame oil; olive oil; corn oil and soybean oil; glycols; such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogen free water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Furthermore, the carrier may take a wide variety of forms depending onthe form of the preparation desired for administration, e.g. oral,nasal, rectal, vaginal, parenteral (including intravenous injections orinfusions). In preparing compositions for oral dosage form any of theusual pharmaceutical media may be employed. Usual pharmaceutical mediainclude, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as for example, suspensions, solutions, emulsions andelixirs); aerosols; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like, in the case of oral solidpreparations (such as for example, powders, capsules, and tablets).

Pharmaceutical compositions comprising the polymorphs of the presentinvention (e.g., Polymorph C) may be formulated to have anyconcentration desired. In some embodiments, the composition isformulated such that it comprises at least a therapeutically effectiveamount. As used herein, “therapeutically effective amount” means thatamount necessary to make a clinically observed improvement in thepatient. In some embodiments, the composition is formulated such that itcomprises an amount that would not cause one or more unwanted sideeffects.

Pharmaceutical compositions include those suitable for oral, sublingual,nasal rectal, vaginal, topical, buccal and parenteral (includingsubcutaneous, intramuscular, and intravenous) administration, althoughthe most suitable route will depend on the nature and severity of thecondition being treated. The compositions may be conveniently presentedin unit dosage form, and prepared by any of the methods well known inthe art of pharmacy. In certain embodiments, the pharmaceuticalcomposition is formulated for oral administration in the form of a pill,capsule, lozenge or tablet. In other embodiments, the pharmaceuticalcomposition is in the form of a suspension.

The compounds provided herein are suitable as an active agent inpharmaceutical compositions that are efficacious particularly fortreating EZH2-associated disorders, especially cancer. Thepharmaceutical composition in various embodiments has a pharmaceuticallyeffective amount of a polymorph of the present invention (e.g.,Polymorph C), along with other pharmaceutically acceptable excipients,carriers, fillers, diluents and the like.

A therapeutically or pharmaceutically “effective amount” is an amount ofa polymorph of the present invention (e.g., Polymorph C), that whenadministered to a patient, ameliorates a symptom of an EZH2-mediateddisease or condition, e.g., prevent the various morphological andsomatic symptoms of an EZH2-mediated cancer. The amount can varydepending on such factors as the size and weight of the subject, thetype of illness, or the particular compound of the invention. The amountof a polymorph of the present invention (e.g., Polymorph C) thatconstitutes an “effective amount” will vary depending on the compound,the disease state and its severity, the age of the patient to betreated, and the like. The effective amount can be determined routinelyby one of ordinary skill in the art having regard to their knowledge andto this disclosure.

The regimen of administration can affect what constitutes apharmaceutically effective amount. A polymorph of the present invention(e.g., Polymorph C), and compositions thereof, can be administered tothe subject either prior to or after the onset of a disease. Further,several divided dosages, as well as staggered dosages can beadministered daily or sequentially, or the dose can be continuouslyinfused, or can be a bolus injection. Further, the dosages can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation. Further, the dosages may beco-administered in combination with other chemotherapeutic agents knownby the skilled artisan.

Methods of Treatment

Polymorphs of the present invention (e.g., Polymorph C) inhibit thehistone methyltransferase activity of EZH2 or a mutant thereof and,accordingly, in one aspect of the invention, certain polymorphsdisclosed herein are candidates for treating, or preventing certainconditions and diseases in which EZH2 plays a role. The presentinvention provides methods for treating conditions and diseases thecourse of which can be influenced by modulating the methylation statusof histones or other proteins, wherein said methylation status ismediated at least in part by the activity of EZH2. Modulation of themethylation status of histones can in turn influence the level ofexpression of target genes activated by methylation, and/or target genessuppressed by methylation. The method includes administering to asubject in need of such treatment, a therapeutically effective amount ofa polymorph of the present invention (e.g., Polymorph C).

Unless otherwise stated, any description of a method of treatmentincludes uses of the polymorphs to provide such treatment or prophylaxisas is described in the specification, as well as uses of the polymorphsto prepare a medicament to treat or prevent such condition. Thetreatment includes treatment of human or non-human animals includingrodents and other disease models.

In still another aspect, this invention relates to a method ofmodulating the activity of the EZH2, the catalytic subunit of the PRC2complex which catalyzes the mono-through tri-methylation of lysine 27 onhistone H3 (H3-K27) in a subject in need thereof. For example, themethod comprises the step of administering to a subject having a cancerexpressing a mutant EZH2 (e.g., a Y641 mutant of EZH2) a therapeuticallyeffective amount of a polymorph described herein, wherein the polymorphinhibits histone methyltransferase activity of EZH2, thereby treatingthe cancer.

For example, the EZH2-mediated cancer is selected from the groupconsisting of follicular lymphoma and diffuse large B-cell lymphoma(DLBCL) of germinal center B cell-like (GCB) subtype. For example, thecancer is lymphoma, leukemia or melanoma. Preferably, the lymphoma isnon-Hodgkin's lymphoma (NHL), follicular lymphoma or diffuse largeB-cell lymphoma. Alternatively, the leukemia is chronic myelogenousleukemia (CML), acute myeloid leukemia, acute lymphocytic leukemia ormixed lineage leukemia.

For example, the EZH2-mediated precancerous condition is myelodysplasticsyndromes (MDS, formerly known as preleukemia).

For example, the EZH2-mediated cancer is a hematological cancer.

The polymorph of the present invention (e.g., Polymorph C) inhibits thehistone methyltransferase activity of EZH2 or a mutant thereof and,accordingly, the present invention also provides methods for treatingconditions and diseases the course of which can be influenced bymodulating the methylation status of histones or other proteins, whereinsaid methylation status is mediated at least in part by the activity ofEZH2. In one aspect of the invention, certain polymorphs disclosedherein are candidates for treating, or preventing certain conditions anddiseases. Modulation of the methylation status of histones can in turninfluence the level of expression of target genes activated bymethylation, and/or target genes suppressed by methylation. The methodincludes administering to a subject in need of such treatment, atherapeutically effective amount of a polymorph of the presentinvention.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refer to a subject having a disorder in whichEZH2-mediated protein methylation plays a part, or a subject having anincreased risk of developing such disorder relative to the population atlarge. A “subject” includes a mammal. The mammal can be e.g., a human orappropriate non-human mammal, such as primate, mouse, rat, dog, cat,cow, horse, goat, camel, sheep or a pig. The subject can also be a birdor fowl. In one embodiment, the mammal is a human. A subject in needthereof can be one who has been previously diagnosed or identified ashaving cancer or a precancerous condition. A subject in need thereof canalso be one who has (e.g., is suffering from) an EZH2-mediated cancer oran EZH2-mediated precancerous condition. Alternatively, a subject inneed thereof can be one who has an increased risk of developing suchdisorder relative to the population at large (i.e., a subject who ispredisposed to developing such disorder relative to the population atlarge). A subject in need thereof can have an EZH2-mediated precancerouscondition. A subject in need thereof can have refractory or resistantEZH2-mediated cancer (i.e., cancer that doesn't respond or hasn't yetresponded to treatment). The subject may be resistant at start oftreatment or may become resistant during treatment. In some embodiments,the subject in need thereof has cancer recurrence following remission onmost recent therapy. In some embodiments, the subject in need thereofreceived and failed all known effective therapies for cancer treatment.In some embodiments, the subject in need thereof received at least oneprior therapy. In a preferred embodiment, the subject has anEZH2-mediated cancer or an EZH2-mediated cancerous condition. Forexample, the EZH2-mediated cancer is lymphoma, leukemia, melanoma, orrhabdomyosarcoma. Preferably, the lymphoma is non-Hodgkin's lymphoma,follicular lymphoma or diffuse large B-cell lymphoma. Alternatively, theleukemia is chronic myelogenous leukemia (CML). The precancerouscondition is myelodysplastic syndromes (MDS, formerly known aspreleukemia).

As used herein, “treating,” “treatment” or “treat” describes themanagement and care of a patient for the purpose of combating a disease,condition, or disorder and includes the administration of a polymorph ofthe present invention (e.g., Polymorph C), to alleviate the symptoms orcomplications of a disease, condition or disorder, or to eliminate thedisease, condition or disorder. The term “treat” can also includetreatment of a cell in vitro or an animal model.

A polymorph of the present invention may also be used to prevent arelevant disease, condition or disorder, or used to identify suitablecandidates for such purposes. As used herein, “preventing,” “prevent,”or “protecting against” describes reducing, ameliorating or eliminatingthe onset of the symptoms or complications of such disease, condition ordisorder.

Point mutations of the EZH2 gene at a single amino acid residue (e.g.,Y641, A677, and A687) of EZH2 have been reported to be linked tolymphoma. More examples of EZH2 mutants and methods of detection ofmutation and methods treatment of mutation-associated disorders aredescribed in, e.g., U.S. Patent Application Publication No. US20130040906, the entire content of which is incorporated herein byreference in its entirety.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, New York (2000); Coligan et al.,Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al.,Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl etal., The Pharmacological Basis of Therapeutics (1975), Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18^(th)edition (1990). These texts can, of course, also be referred to inmaking or using an aspect of the invention.

All percentages and ratios used herein, unless otherwise indicated, areby weight (i.e., weight by weight or wt/wt). Other features andadvantages of the present invention are apparent from the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

EXAMPLES X-Ray Powder Diffraction

XRPD for all samples was taken on a Rigaku MultiFlex (Target: Cu; Tubevoltage: 40 kV; Tube current: 30 mA, at room temperature (about 25° C.),and at 30% relative humidity (RH)).

Differential Scanning Calorimetry

DSC for all samples was taken on a Mettler-Toledo DSC 1/700 (Runconditions: Initial temperature 35° C., Final temp 325-350° C., Heatingrate 10-30° C./min).

Dynamic Vapor Sorption

DVS was measured on a VTI Model SGA-100 system. Measurement method: Therelative humidity (RH) was changed in a controlled fashion, in 5% stepsfrom 5.0% to 95.0% then back to 5.0% using the gravimetric vaporsorption system, and the weight percentage change (wt%) of the sample ateach stage was measured.

Synthesis of Compound I

5-bromo-2-methyl-3-nitrobenzoic acid: To a stirred solution of2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in conc. H₂SO₄ (400 mL),1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) wasadded in a portion wise manner at room temperature and the reactionmixture was then stirred at room temperature for 5 h. The reactionmixture was poured onto ice cold water, the precipitated solid wasfiltered off, washed with water and dried under vacuum to afford thedesired compound as a solid (140 g, 98%). The isolated compound wastaken directly into the next step. ¹H NMR (DMSO-d₆, 400 MHz) δ 8.31 (s,1H), 8.17 (s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate: To a stirred solution of5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8L) atroom temperature was added sodium carbonate (468 g, 4415 mmol) followedby addition of methyl iodide (626.6 g, 4415 mmol). The resultingreaction mixture was heated at 60° C. for 8 h. After completion(monitored by TLC), the reaction mixture was filtered (to remove sodiumcarbonate) and washed with ethyl acetate (1L×3). The combined filtratewas washed with water (3L×5) and the aqueous phase was back extractedwith ethyl acetate (1L×3). The combined organic layers were dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to afford the title compound as a solid (290 g, 97% yield). Theisolated compound was taken directly into the next step. ¹H NMR (CDCl₃,400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate : To a stirred solution ofmethyl 5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol(1.5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolvedin 1.5L water). The resulting mixture was stirred at 80° C. to whichiron powder (472 g, 8451 mmol) was added in a portion wise manner. Theresulting reaction mixture was heated at 80° C. for 12 h. Uponcompletion as determined by TLC, the reaction mixture was hot filteredover celite® and the celite bed was washed with methanol (5L) followedby washing with 30% MeOH in DCM (5L). The combined filtrate wasconcentrated in-vacuo, the residue obtained was diluted with aqueoussodium bicarbonate solution (2L) and extracted with ethyl acetate(5L×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to afford thetitle compound as a solid (220 g, 85%). The compound was taken directlyinto the next step. ¹H NMR (CDCl₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s,1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate :A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate(455.8 g, 1.87 mol), 1,2-Dichloroethane (4.56 L), and acetic acid (535ml, 9.34 mol). To the mixture were added dihydro-2H-pyran-4(3H)-one (280g, 2.80 mol) and sodium triacetoxyborohydride (594 g, 2.80 mol)maintaining the internal temperature below 40° C. The mixture wasstirred at 25° C. for 2.5 h and then the reaction was quenched with asolution of sodium hydroxide (448 g, 11.20 mol) in water (5.61 L). Afterstirring for 20 minutes at ambient temperature, the organic layer wasseparated and the aqueous layer was extracted with ethyl acetate (3.65L). The organic layers were combined, washed with brine (1.5 L), andconcentrated under vacuum.

The residue was treated with ethyl acetate (1.8 L) and heated to 65-70°C. The mixture was stirred at 65-70° C. for 15 minutes to give a clearsolution and then treated with n-heptane (7.3 L) maintaining thetemperature between 60-70° C. Once the heptane was completely added tothe solution, the mixture was held at 65-70° C. for 15 minutes and thenallowed to cool to 18-22° C. over 3 h. The resulting suspension wasstirred at 18-22° C. for 4 h, cooled to 0-5° C. over 1 h, and held at0-5° C. for 2 h. The precipitate was filtered, washed twice withn-heptane (1.4 L), and dried under vacuum to give the title compound(540 g, 88%).

Methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate : To a stirred solution of methyl5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g,42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g,85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reactionmixture was stirred at room temperature for 15 minutes. The mixture wascooled to 0° C. and sodium triacetoxyborohydride (27 g, 128 mmol) wasadded. The reaction mixture was stirred at room temperature for 3 hours.Upon completion of the reaction as determined by TLC, aqueous sodiumbicarbonate solution was added to the reaction mixture until a pH 7-8was obtained, the organic phase was separated and the aqueous phase wasextracted with ethyl acetate. The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure. The crude compound was purified by column chromatography(100-200 mesh silica gel) eluting with ethyl acetate: hexane to affordthe desired compound as a viscous liquid (14 g, 93%). ¹H NMR (DMSO-d₆,400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H),2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H),1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Methyl5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4%(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylate: A mixture of methyl5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (580g, 1.63 mol),4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine (592g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 mol),and water (771 ml) was degassed by bubbling nitrogen through the mixtureat 20° C. for 20 minutes and treated withtetrakis(triphenylphosphine)palladium(0) (14.11 g, 12.21 mmol). Theresulting mixture was degassed for an additional 20 minutes and thenheated to 87-89° C. for 17 h. After cooling to 20° C., the mixture wasdiluted with ethyl acetate (5.80 L) and a solution of(R)-2-Amino-3-mercaptopropionic acid (232 g) in water (2.320 L). Afterstirring for 1 h at 20° C., the organic layer was separated and washedagain with a solution of (R)-2-Amino-3-mercaptopropionic acid (232 g) inwater (2.320 L). The aqueous layers were combined and extracted withethyl acetate (5.80 L). The organic layers were combined, washed with asolution of sodium hydroxide (93 g) in water (2.32 L), and concentratedunder vacuum at 35° C. to give the title compound as an orange oil (1.21kg, 164% yield).

5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4%(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylicacid: Methyl5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylate(69.0 g, 152.5 mmol) (based on the theoretical yield from the previousstep) was suspended in ethanol (380 mL) and treated with a solution ofsodium hydroxide (24.84 g, 621.0 mmol) in water (207 mL). The mixturewas stirred at 40° C. for 18 h. After cooling to 0-5° C., the mixturewas neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL)maintaining the temperature below 25° C. Then, the mixture was extractedtwice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL).The organic layers were combined and concentrated under vacuum to give acrude product as a yellow solid (127g).

The crude product was dissolved in 2-methyltetrahydrofuran (656 mL) at70° C. and then treated with IPA (828 mL). The mixture was allowed tocool to rt over 3-4 h and then stirred overnight at rt. The precipitatewas filtered, washed twice with IPA (207 mL), and dried under vacuum togive the title compound as an off white solid (53.54 g, 80%).

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyhtetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(Compound I): A mixture of5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxylicacid (540 g, 1.23 mol) and3-(aminomethyl)-4,6-dimethyl-dihydro-pyridin-2(1H)-one hydrochloride(279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated withtriethylamine (223 ml, 1.60 mol). The mixture was stirred at 25° C. for30 min and treated with EDC-HCl (354 g, 1.85 mol) and HOBT hydrate (283g, 1.85 mol). The reaction mixture was stirred at rt for 16 h. Afteraddition of triethylamine (292 ml, 2.09 mol), the mixture was cooled to15° C., diluted with water (10.1 L) maintaining the temperature below30° C., and stirred at 19-25° C. for 4 h. The resulting precipitate wasfiltered, washed twice with water (2.70 L), and dried under vacuum togive a crude product (695 g, wt-wt analysis=78%).

For the further purification of the product, recrystallization wasconducted. A crude product (20.00 g, 34.92 mmol) was suspended in amixture of ethanol (190 ml) and water (10.00 ml) and heated to 75° C.until a clear solution was obtained. The solution was allowed to cool tort overnight. The precipitate was filtered, washed twice with a mixtureof ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35°C. to give the title compound as an off white solid (14.0 g, 70%recovery from the crude and 90% yield based on wt-wt assay).

Preparation of Polymorph C

4-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyhtetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumchloride

A suspension ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide(10.0 g, 17.46 mmol) in ethanol (70.0 ml) was heated to 70° C. (bath)and treated with cone HCl (1.455 ml, 17.46 mmol). The mixture wasstirred at 70° C. for 20 min and then treated with ethyl acetate (140.0ml). The resulting mixture was stirred at 70° C. for 30 min and slowlycooled to room temperature over 20 h. The resulting precipitate wasfiltered, washed with ethyl acetate (20 mL) and dried over N₂ purge for20 h to give Polymorph C (6.17 g, 63%).

Preparation of Polymorph A

4-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumchloride (Polymorph A)

A suspension ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl -4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide (100 mg, 0.18 mmol) ina mixture of ethanol (0.2 mL) and water (0.1 mL) was heated to 80° C.(bath) and treated with conc. HCl (0.29 mL, 3.49 mmol). The resultingclear solution was treated with ethanol (1 ml) at 80° C. (bath), andstirred at 40° C. (bath) for 30 min and at rt for 16 h. The resultingprecipitate was filtered, washed with ethanol (1 mL) and dried over N₂purge to give a crude title compound (60 mg).

The crude HCl salt was treated with ethyl acetate (1 mL), heated to 80°C. (bath), and treated with methanol (0.15 mL) to give a clear solution.The mixture was stirred at ambient temperature for 16 h. The precipitatewas filtered, washed with ethyl acetate, and dried over N₂ purge to givethe title compound of Polymorph A (54 mg, 51%).

Preparation of Polymorph B

4-((3′-(((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[1,1′-biphenyl]-4-yl)methyl)morpholin-4-iumchloride (Polymorph B)

N-((4, 6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide (2.0 g, 3.49 mmol) wassuspended in a mixture of methanol (2.65 mL) and ethyl acetate (2.65mL), and heated to 60° C. (bath). The mixture was treated with conc. HCl(0.29 mL, 3.49 mmol). The resulting clear dark solution was treated withethyl acetate (10 ml), stirred at 60° C. (bath) for 10 min, and slowlycooled to rt over 20 h. The resulting precipitate was filtered, washedtwice with ethyl acetate (5 mL) and dried over N₂ purge for 4 h to givea crude title compound (2.05 g, 96%).

Polymorph B was Prepared by Two Methods:

a) 200 mg of the crude HCl salt was treated with acetonitrile (3 mL),heated to 70° C. (bath), and treated with water (0.3 mL) to give a clearsolution. The mixture was stirred at 70° C. (bath) for 10 min and slowlycooled to rt over 20 h. The precipitate was filtered, washed withacetonitrile, and dried under vacuum for 4 h to give the title compoundof Polymorph B (160 mg, 80%).

b) 200 mg of the crude HCl salt was treated with acetone (3 mL), heatedto 70° C. (bath), and treated with water (0.45 mL) to give a clearsolution. The mixture was stirred at 70° C. (bath) for 10 min and slowlycooled to rt over 20 h. The precipitate was filtered, washed withacetonitrile, and dried under vacuum for 4 h to give the title compoundof Polymorph B (152 mg, 76%).

Tables

TABLE 1 Polymorph A 2-Theta 11.22 12.0 13.116 13.418 13.899 17.02618.032 18.32 19.399 20.199 21.84 22.499 23.238 24.363 24.7 24.958 30.55730.879

TABLE 2 Polymorph B 2-Theta 8.438 10.18 10.74 13.318 13.541 13.76216.443 17.219 17.78 18.419 20.182 20.421 20.839 21.958 23.725 24.15925.498 26.863

TABLE 3 Polymorph C 2-Theta 10.083 10.940 16.583 17.124 17.534 18.34018.662 20.500 21.143 21.917 22.219 23.460 26.222 26.596 27.722 30.299

TABLE 4 Polymorph Polymorph A Polymorph B Polymorph C Onset temperature190 ± 5° C. 105 ± 5° C. 228 ± 5° C. (° C.) (primary endotherm)

Characteristics of Polymorph Forms

Three solid crystalline forms of Compound I hydrochloride were preparedand characterized. These forms are identified herein as Polymorph A,Polymorph B and Polymorph C. Among them, Polymorph C had the mostadvantageous physicochemical properties in terms of stability (cf. FIG.4) and hygroscopicity (cf. FIGS. 5 and 6). The formation of Polymorph Cas described herein is also advantageous in that it results in a form ofCompound I HCl that is substantially free of amorphous Compound I (orits amorphous mono- or multi-HCl forms).

As shown in FIG. 7, DSC data of Polymorph A indicates some degree ofnon-crystallinity with an endotherm at 190.5° C. Also, dynamic vaporsorption (DVS) data for Polymorph A was obtained and found to show somehygroscopicity: between 4-6% weight gain was observed at 75% relativehumidity (RH) at 25° C. (FIG. 6).

Surprisingly, Polymorph C was found to be highly crystalline and stable(with the highest endotherm of the three polymorphic forms discussedherein; see FIG. 4) and non-hygroscopic (FIG. 5).

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1.-6. (canceled)
 7. A method of treating cancer comprising administeringto a subject in need thereof a therapeutically effective amount of asolid crystalline form ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide,or a salt thereof, wherein the solid crystalline form exhibits an X-raypowder diffraction pattern having two or more peaks expressed in degrees2-theta (+/−0.2), selected from the group consisting of 8.438, 10.083,10.18, 10.74, 10.940, 11.22, 12.0, 13.116, 13.318, 13.418, 13.541,13.762, 13.899, 16.443, 16.583, 17.026, 17.124, 17.219, 17.53, 17.78,18.032, 18.32, 18.340, 18.419, 18.66, 19.399, 20.182, 20.199, 20.421,20.500, 20.839, 21.14, 21.84, 21.917, 21.958, 22.22, 22.499, 23.238,23.46, 23.725, 24.159, 24.363, 24.7, 24.958, 25.498, 26.222, 26.596,26.863, 27.72, 30.30, 30.557, and 30.879.
 8. The method of claim 7,wherein the cancer is EZH2 mediated.
 9. The method of claim 7, whereinthe solid crystalline form exhibits an X-ray powder diffraction patternhaving two or more characteristic peaks expressed in degrees 2-theta(+/−0.2), selected from the group consisting of 17.53, 18.66, 21.14,22.22, 23.46, 27.72 and 30.30.
 10. The method of claim 7, wherein thesolid crystalline form exhibits an X-ray powder diffraction patternhaving three or more characteristic peaks expressed in degrees 2-theta(+/−0.2), selected from the group consisting of 17.53, 18.66, 21.14,22.22, 23.46, 27.72 and 30.30.
 11. The method of claim 7, wherein thesolid crystalline form exhibits an X-ray powder diffraction patternhaving characteristic peaks expressed in degrees 2-theta (+/−0.2) at17.53, 21.14, 23.46, and 27.72.
 12. The method of claim 7, wherein thesolid crystalline form exhibits a differential scanning calorimetrythermogram showing a primary endotherm expressed in units of ° C. at atemperature of 190+/−5° C., 105+/−5° C., or 228+/−5° C. 13.-19.(canceled)
 20. The method of claim 7, wherein the solid crystalline formis a hydrochloride salt.
 21. A polymorph ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidemonohydrochloride salt.
 22. The polymorph of claim 21, wherein thepolymorph exhibits an X-ray powder diffraction pattern having two ormore peaks expressed in degrees 2-theta (+/−0.2), selected from thegroup consisting of 8.438, 10.083, 10.18, 10.74, 10.940, 11.22, 12.0,13.116, 13.318, 13.418, 13.541, 13.762, 13.899, 16.443, 16.583, 17.026,17.124, 17.219, 17.53, 17.78, 18.032, 18.32, 18.340, 18.419, 18.66,19.399, 20.182, 20.199, 20.421, 20.500, 20.839, 21.14, 21.84, 21.917,21.958, 22.22, 22.499, 23.238, 23.46, 23.725, 24.159, 24.363, 24.7,24.958, 25.498, 26.222, 26.596, 26.863, 27.72, 30.30, 30.557, and30.879.
 23. The polymorph of claim 21, wherein the polymorph exhibits anX-ray powder diffraction pattern having two or more characteristic peaksexpressed in degrees 2-theta (+/−0.2), selected from the groupconsisting of 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and 30.30. 24.The polymorph of claim 21, wherein the polymorph exhibits an X-raypowder diffraction pattern having three or more characteristic peaksexpressed in degrees 2-theta (+/−0.2), selected from the groupconsisting of 17.53, 18.66, 21.14, 22.22, 23.46, 27.72 and 30.30. 25.The polymorph of claim 21, wherein the polymorph exhibits an X-raypowder diffraction pattern having characteristic peaks expressed indegrees 2-theta (+/−0.2) at 17.53, 21.14, 23.46, and 27.72.
 26. Thepolymorph of claim 21, wherein the polymorph exhibits a differentialscanning calorimetry thermogram showing a primary endotherm expressed inunits of ° C. at a temperature of 190+/−5° C., 105+/−5° C., or 228+/−5°C.
 27. The polymorph of claim 21, wherein the polymorph exhibits anX-ray powder diffraction pattern having two or more characteristic peaksexpressed in degrees 2-theta (+/−0.2), selected from the groupconsisting of 11.22, 12.0, 13.116, 18.032, 19.399, 22.499, 24.363, 24.7,24.958, 30.557, and 30.879.
 28. The polymorph of claim 21, wherein thepolymorph exhibits an X-ray powder diffraction pattern having three ormore characteristic peaks expressed in degrees 2-theta (+/−0.2),selected from the group consisting of 11.22, 12.0, 13.116, 18.032,19.399, 22.499, 24.363, 24.7, 24.958, 30.557, and 30.879.
 29. Thepolymorph of claim 21, wherein the polymorph exhibits an X-ray powderdiffraction pattern having two or more characteristic peaks expressed indegrees 2-theta (+/−0.2), selected from the group consisting of 8.438,13.318, 17.78, 23.725,
 24. 159, 25.498 and 26.863.
 30. The polymorph ofclaim 21, wherein the polymorph exhibits an X-ray powder diffractionpattern having three or more characteristic peaks expressed in degrees2-theta (+/−0.2), selected from the group consisting of 8.438, 13.318,17.78, 23.725,
 24. 159, 25.498 and 26.863.
 31. The polymorph of claim21, wherein the polymorph exhibits an X-ray powder diffraction patternhaving two or more peaks expressed in degrees 2-theta (+/−0.2) selectedfrom the group consisting of about 10.083, 10.940, 13.418, 13.899,16.583, 17.124, 17.53, 18.340, 18.66, 20.199, 20.500, 20.839, 21.917,and 23.46.
 32. The polymorph of claim 21, wherein the polymorph exhibitsan X-ray powder diffraction pattern having two or more peaks expressedin degrees 2-theta (+/−0.2) selected from the group consisting of10.940, 13.418, 13.899, 16.583, 17.124, 18.340, and 20.199.
 33. Apharmaceutical composition comprising a polymorph of claim 21, and apharmaceutically acceptable carrier or diluent.